B. Abstract and Specific Aims The goal of our Nanomedicine Development Center has been to use outside-the-box, high-risk, high-payoff and highly interdisciplinary approaches and methodologies to obtain a quantitative understanding of cellular protein folding nanomachines such as mammalian TRiC and the archaeal Mm-cpn chaperonins. Our objective is to re-engineer them for biomedical applications. Central to our approach is the iterative application of scientific, engineering and clinical principles. Our ultimate goal is to harness our knowledge of protein folding machines to create novel therapeutic agents for inhibiting and/or promoting the folding bf selected proteins whose misfolding or aggregation are associated with human diseases. At the inception of our Center, we assembled a team of 15 investigators from 6 institutions with expertise in chaperone biochemistry, protein folding, electron cryomicrosopy, computer simulation and design. X-ray crystallography, single-molecule imaging and trapping, and clinical research. Through the translational supplement available in the mid-course of the current grant cycle, we recruited 8 medical investigators who are specialists in different disease targets, all of which are tied to protein misfolding or aggregation. In the past 5 years, we have made substantial advances in the development of technologies for characterizing chaperonins and other cellular nanomachines in unprecedented detail and in identifying protein folding, misfolding and aggregation disease targets to pave the way for the development of novel nanomedicine therapeutics. In this renewal, we seek to continue the interdisciplinary collaborations among 17 basic and physician scientists from 8 institutions to implement the second phase of our NDC We aim to bring our basic research closer to translational reality in alleviating the deleterious effects of misfolded or aggregated proteins in human diseases including Von Hippel Landau syndrome, stroke and ischemic injury, cystic fibrosis, Huntingtons disease, cataracts, and Alzheimer's disease. Our goals are (i) to utilize the deep insights we have gained into TRiC and Mm-cpn structure and function in the past few years to engineer novel modified forms of chaperonins, chaperonin subunits or peptides for diagnostic and therapeufic purposes; (ii) to design and screen novel adaptor molecules that expand or modify chaperonin-substrate recognition, alter protein folding and prevent protein aggregation; and (iii) to use the chaperonin nanomachine as a novel target for the identification of small molecules from existing NIH libraries that can be developed for therapeufic use. The main philosophy of the Center is to continue our highly interdisciplinary strategy and collaboration to develop cutting-edge methodologies and therapeufic solutions. Our approach consists of 4 major aims aligned with stages in the therapeutics discovery process: Aim 1: Identify and validate therapeutic strategies for six selected disease targets Aim 2: Design and screen novel therapeutic agents Aim 3: Assess molecular mechanism of therapeufic action Aim 4: Perform pre-clinical model testing. Although our team's nucleus was a group of basic scientists, we recognize the need to scale down the level of basic science research in order to accomplish the NDC goal, which is to transform our research into medical applications as quickly as possible. Nevertheless, we plan to maintain sufficient basic science investigation to validate and to measure the functional properties of the newly discovered therapeutics in the context of the cell or cell-like environment Such validation is critical to guide and adjust the design process, thus improving the quality of the engineered products finally administered in vivo. After an initial evaluation period, we will select among the initial disease targets and concentrate on a reduced number of targets in the third year of this grant cycle. This will enable us to better develop and optimize therapeutics that will lead to clinical trials. In preparation for the later phases of the translational process, we will engage in a dialogue and collaborations with necessary specialists both in academia and in industry; including bioengineers, pharmacologists and toxicologists so that our intellectual concepts and discoveries can be brought to human trials promptly.